LRRC8/VRAC volume-regulated anion channels are crucial for hearing

Hearing crucially depends on cochlear ion homeostasis as evident from deafness elicited by mutations in various genes encoding cation or anion channels and transporters. Ablation of ClC‑K/barttin chloride channels causes deafness by interfering with the positive electrical potential of the endolymph, but roles of other anion channels in the inner ear have not been studied. Here we report the intracochlear distribution of all five LRRC8 subunits of VRAC, a volume-regulated anion channel that transports chloride, metabolites, and drugs such as the ototoxic anti-cancer drug cisplatin, and explore its physiological role by ablating its subunits. Sensory hair cells express all LRRC8 isoforms, whereas only LRRC8A, D and E were found in the potassium-secreting epithelium of the stria vascularis. Cochlear disruption of the essential LRRC8A subunit, or combined ablation of LRRC8D and E, resulted in cochlear degeneration and congenital deafness of Lrrc8a-/- mice. It was associated with a progressive degeneration of the organ of Corti and its innervating spiral ganglion. Like disruption of ClC-K/barttin, loss of VRAC severely reduced the endocochlear potential. However, the mechanism underlying this reduction seems different. Disruption of VRAC, but not ClC-K/barttin, led to an almost complete loss of Kir4.1 (KCNJ10), a strial K+ channel crucial for the generation of the endocochlear potential. The strong downregulation of Kir4.1 might be secondary to a loss of VRAC-mediated transport of metabolites regulating inner ear redox potential such as glutathione. Our study extends the knowledge of the role of cochlear ion transport in hearing and ototoxicity.

In in 20-week-old Sox10-Cre; R26R mice, X-Gal staining reveals Cre-recombinase expression in the organ of Corti, stria vascularis, spiral ganglion, Reissner`s membrane, and cells in outer sulcus and spiral prominence.In the crista, saccule (S) and utricle (U), the β-galactosidase is expressed in the sensory epithelia and vestibular membranes.B. Specificity of cell-type specific Lrrc8a disruption in Sox10-Cre; Lrrc8a lox HA/lox HA mice (P12).HA-tagged LRRC8A could not be detected in the stria vascularis (SV) and organ of Corti (OC).Vasculature in the spiral limbus (SLB), stria vascularis (SV) and the spiral ligament (SL), few cells in Reissner's membrane (RM), type II and type IV fibrocytes and tympanic border cells (TBC) retained an HA signal.I-V fibrocytes type I to V; SP spiral prominence and outer sulcus region; IHC inner hair cells; OHC outer hair cells.

Figure S1 .
Figure S1.LRRC8A subunit expression in cochlea.A. X-Gal stained cochlear section from 13-week-old Lrrc8a +/lacZ mouse.The Lrrc8a promotor is active in cells of the spiral ganglion (SG), organ of Corti (OC), Reissner`s membrane (RM), stria vascularis (SV), spiral prominence and outer sulcus region (SP), and in the spiral limbus (SLB).No labeling was detected in fibrocytes and bone.B. LRRC8A is expressed in strial cells, labelled with marginal cell marker KCNQ1.23-week-old Lrrc8a lox HA/lox HA mouse.Images are collected from at least 2 different mice.

Figure S2 .
Figure S2.LRRC8A expression in vestibular organ.A-B.LRRC8A was detected by HA-epitope labeling in vestibular section of an 11-week-old Lrrc8a lox HA/lox HA mouse and compared to control WT mice.LRRC8A is present in the sensory epithelium (SE) and fibrocytes of connective tissue (CT) in the crista (A) and the utricle (B), and also in dark cells (DC) and the wall of ampulla (WA).C. LRRC8A expression as indicated by X-Gal staining of vestibular sections from 13-week-old Lrrc8a +/lacZ and WT control mice.Labeling is observed in the sensory epithelium (SE), transitional cells (TC) and dark cells (DC) of the crista (left panel), as well as in the sensory epithelia (SE) and the epithelium of the membrane labyrinth (E) in utricle (U) and saccule (S) (right panel).Labeling is also observed in the cells of the vestibular ganglion (VG).D. Higher magnification showing HA-staining of hair cells in the crista of a 11-week-old Lrrc8a lox HA/lox HA mouse.Punctate staining of the outer membrane and the cytoplasm.LRRC8A is almost absent from calyx synapses identified by KCNQ4 K + -channels present in calyx terminals ensheathing type I hair cells (72).Images are collected from at least two different mice.

Figure S3 .
Figure S3.Expression of LRRC8B-E in the vestibular organ.A. X-Gal stained vestibular sections from 6week-old Lrrc8b +/lacZ and control WT mice.β-Galactosidase is expressed in the sensory epithelia (SE) and fibrocytes (F) in the vestibular organ.No staining was detected in the epithelium of the membranous labyrinth (E) and dark cells (DC) in the crista.S, saccule; U, utricle.B. LRRC8C is weakly expressed in vestibular hair cells, whose calyx terminals are labelled with KCNQ4.Blood vessels strongly express

Figure S4 .
Figure S4.Specificity of cell-type specific Lrrc8a disruption under Sox10-Cre promotor.A. Expression pattern of Cre-recombinase inner ear in Sox10-Cre mice crossed to reporter mice.In in 20-week-old Sox10-Cre; R26R mice, X-Gal staining reveals Cre-recombinase expression in the organ of Corti, stria vascularis, spiral ganglion, Reissner`s membrane, and cells in outer sulcus and spiral prominence.In the crista, saccule (S) and utricle (U), the β-galactosidase is expressed in the sensory epithelia and vestibular membranes.B. Specificity of cell-type specific Lrrc8a disruption in Sox10-Cre; Lrrc8a lox HA/lox HA mice (P12).HA-tagged LRRC8A could not be detected in the stria vascularis (SV) and organ of Corti (OC).Vasculature

Figure S5 .
Figure S5.Changes in levels of ion transport proteins secondary to LRRC8A knockout.A. Quantification of transport proteins levels from immunohistochemistry. Kir4.1 and pendrin protein levels, but not those of other transporters of the stria vascularis (KCNQ1, ClC-K, NKCC1, barrtin, Na,K-ATPase) and spiral ligament (KCC3), are reduced in 16-20-days-old Sox10-Cre; Lrrc8a lox/lox compared to wild-type mice.Average gray values were divided by a mean value among wild-types, each point represent an average value across different inner ear slices of one mouse.Truncated violin plots, median is represented as a solid line, quartiles as broken line.Mann-Whitney test for Kir4.1, unpaired t test for pendrin.KCNQ1: WT N=7, KO N=8; Na,K-ATPase: WT and KO N=7, NKCC1: WT N=6, KO N=7; ClC-K1: WT N=7, KO N=8; Barrtin: WT and KO N=7; KCC3: WT and KO N=8; Kir4.1:WT N=6, KO N=7; Pendrin: WT N=7, KO N=8.B. Reduced expression of pendrin in the spiral ligament of 2-week-old Sox10-Cre; Lrrc8a lox/lox mice.C. Kir4.1 and pendrin mRNA levels are not changed in stria vascularis and spiral ligament of 1-year-old Sox10-Cre; Lrrc8a lox/lox mice.Data is shown as a fold change relative to the mean across all WT.Stria vascularis and spiral ligament were separated from the spiral ganglion, spiral limbus and organ of Corti; the purity of preparation was controlled through mRNA presence of strial marginal cell marker KCNQ1 and outer hair cell marker KCNQ4.Data is not significantly different (unpaired t test).N = 5 for both WT and KO.